Polydatin Prevents Electron Transport Chain Dysfunction and ROS Overproduction Paralleled by an Improvement in Lipid Peroxidation and Cardiolipin Levels in Iron-Overloaded Rat Liver Mitochondria

Abstract

Increased intramitochondrial free iron is a key feature of various liver diseases, leading to oxidative stress, mitochondrial dysfunction, and liver damage. Polydatin is a polyphenol with a hepatoprotective effect, which has been attributed to its ability to enhance mitochondrial oxidative metabolism and antioxidant defenses, thereby inhibiting reactive oxygen species (ROS) dependent cellular damage processes and liver diseases. However, it has not been explored whether polydatin is able to exert its effects by protecting the phospholipid cardiolipin against damage from excess iron. Cardiolipin maintains the integrity and function of electron transport chain (ETC) complexes and keeps cytochrome c bound to mitochondria, avoiding uncontrolled apoptosis. Therefore, the effect of polydatin on oxidative lipid damage, ETC activity, cytochrome levels, and ROS production was explored in iron-exposed rat liver mitochondria. Fe²⁺ increased lipid peroxidation, decreased cardiolipin and cytochromes c + c₁ and aa₃ levels, inhibited ETC complex activities, and dramatically increased ROS production. Preincubation with polydatin prevented all these effects to a variable degree. These results suggest that the hepatoprotective mechanism of polydatin involves the attenuation of free radical production by iron, which enhances cardiolipin levels by counteracting membrane lipid peroxidation. This prevents the loss of cytochromes, improves ETC function, and decreases mitochondrial ROS production.

Keywords: Fe2+; cytochromes; free radicals; hydroxyl radical; iron overload; liver disease; mitochondrial function; oxidative stress; piceid; respiratory chain.

Figure 1

Effect of pretreatment with 100 µM polydatin (PD) or 5 µM butylated hydroxytoluene (BHT) on lipid peroxidation levels in mitochondria exposed to 50 µM ferrous iron (Fe). Results are expressed as the mean ± standard error of n ≥ 3. * p < 0.05 vs. CTRL and ^§ p < 0.05 vs. Fe. (one-way ANOVA, Holm–Sidak post hoc test, p < 0.05).

Figure 2

Effect of pretreatment with 100 µM polydatin (PD) or 5 µM butylated hydroxytoluene (BHT) on cardiolipin levels in mitochondria exposed to 50 µM ferrous iron (Fe). Results are expressed as the mean ± standard error of n ≥ 4. * p < 0.05 vs. CTRL, ^§ p < 0.05 vs. Fe and ^¶ p < 0.05 vs. PD + Fe (one-way ANOVA, Holm–Sidak post hoc test, p < 0.05).

Figure 3

Effect of pretreatment with 100 µM polydatin (PD) or 5 µM butylated hydroxytoluene (BHT) on cytochrome levels in mitochondria exposed to 50 µM ferrous iron (Fe): (a) representative cytochrome differential absorption spectra. Dotted lines indicate the absorption maximum for cytochromes c + c₁ (550 nm) and cytochromes aa₃ (600 nm); (b) quantification of cytochrome c + c₁ levels; (c): quantification of cytochrome aa₃ levels. Results are expressed in (b,c) as the mean ± standard error of n = 4. * p < 0.05 vs. CTRL and ^§ p < 0.05 vs. Fe (one-way ANOVA, Holm–Sidak post hoc test, p < 0.05).

Figure 4

Effect of pretreatment with polydatin (PD) or butylated hydroxytoluene (BHT) on the activity of ETC complexes of mitochondria exposed to Fe²⁺ (Fe): (a) complex I activity (50 µM PD, 2.5 µM BHT, 25 µM Fe²⁺); (b) complex II activity (100 µM PD, 5 µM BHT, 50 µM Fe²⁺); (c) complex III activity (100 µM PD, 5 µM BHT, 50 µM Fe²⁺); (d) complex IV activity (200 µM PD, 5 µM BHT, 100 µM Fe²⁺). Results are expressed as mean ± standard error of n ≥ 3. * p < 0.05 vs. CTRL and ^§ p < 0.05 vs. Fe (one-way ANOVA, Holm–Sidak post hoc test, except for (c), where the Student–Newman–Keuls method was used, p < 0.05).

Figure 5

Effect of pretreatment with 50 µM polydatin (PD) on ROS levels of mitochondria exposed to 25 µM ferrous iron (Fe). Mitochondria were incubated with glutamate–malate (G + M; open bars) to stimulate ROS production and antimycin A (AA; diagonal striped bars) to stimulate maximal ROS production in the ETC. Results are expressed as the mean ± standard error of n ≥ 5. * p < 0.05 vs. CTRL and ^§ p < 0.05 vs. Fe (one-way ANOVA, Holm–Sidak post hoc test, p < 0.05).